Antibody-resin coupling apparatus and methods

ABSTRACT

An antibody-resin coupling apparatus quickly and efficiently activates resin beads and couples them to antibodies, while preventing breakdown and crosslinking of the beads, thereby improving downstream column purification processes, extending the usable life of the resin beads, and increasing molecule capture efficiency of the resultant resin-antibody complexes, to allow improved isolation and purification of factor VIII molecules or other drug compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of, and priority to, U.S.Application No. 62/541,601 filed on Aug. 4, 2017, the contents of whichare incorporated by reference.

FIELD OF THE INVENTION

The present disclosure relates to devices and methods for activatingresins and coupling antibodies to the resin.

BACKGROUND

Hemophilia is an inherited bleeding disorder that prevents blood fromclotting normally and is characterized by hemorrhages, which may occurspontaneously or after minor trauma. It is often associated with adeficiency of an essential blood-clotting protein, factor VIII, alsoknown as anti-hemophilic factor (AHF). In humans, factor VIII is encodedby the F8 gene. Defects in this gene result in hemophilia A (“classic”hemophilia), a recessive X-linked coagulation disorder that results inprolonged plasma clotting time. Another type of hemophilia is acquiredhemophilia A (AHA), which occurs in patients who have normal F8 genesbut develop autoantibodies that inhibit factor VIII, creating afunctional deficiency that impedes coagulation.

Providing intravenous factor VIII to patients with hemophilia cantemporarily improve clotting. Many treatments for hemophilia such as thehemophilia treatments sold under the trademarks RECOMBINATE, ADVATE,ADYNOVATE, HEMOFIL, and OBIZUR, available from Shire Plc (Lexington,Mass.), involve recombinant factor VIII proteins synthesized andpurified in the lab.

However, a major limiting factor in production of such treatments is theability to isolate and purify the factor VIII molecules. One method isto use antibodies of factor VIII to capture the molecules in a column.Antibodies are coupled to a resin and loaded into a column, and mediacontaining expressed factor VIII molecules is passed therethrough.

This purification process requires high precision to properly manipulatethe resin. It can be extremely costly to perform, because the resin isexpensive and can be easily damaged or wasted if not correctlyprocessed. When the resin is not adequately coupled to the antibodies,cross-linking may occur between resin beads. Also the beads may breakdown if the resin is not carefully handled. Broken down or crosslinkedresin beads can clog the column, creating excessive back pressure andcausing the extraction process to fail. Accordingly, the difficultieswith resin handling procedures and the limitations of resin-antibodycoupling technology make the manufacturing process for hemophilia drugsexpensive and complicated.

SUMMARY

The apparatus described herein efficiently activates resin beads andcouples them to antibodies, while preventing breakdown and crosslinkingof the beads, thereby improving downstream column purificationprocesses, extending the usable life of the resin beads, and increasingmolecule capture efficiency of the resultant resin-antibody complexes,to allow improved isolation and purification of a wide variety ofbiological compounds, including factor VIII molecules.

Although the apparatus is generally described herein with reference toits use in FVIII purification, it should be understood that theapparatus and related methods are useful for coupling antibodies of alltypes to porous resins. The inventions of the present disclosure can beapplied to other affinity resins, particularly for other enzymereplacement therapy products or any other drug or biomolecule ofinterest. It is applicable to crosslinking chemistries between protein-or amine-containing ligands and a resin support. The resin may beagarose, glass, or other known porous resins of various densities. Theantibodies used may be either polyclonal or monoclonal. Although thedevice is primarily described herein as being useful for couplingantibodies to resin, but the invention is compatible with any chemistrywherein hydroxyl groups are coupled to amino groups. It should beunderstood that the invention is useful with any known antibody that canbe used for affinity chromatography purification of any biologicalcompound, including peptides, nucleic acids, carbohydrates, and anyligand with amino groups.

The apparatus is generally a mixing device, which includes a vessel withinlets for introducing resin beads and various fluids, a dispersion tubefor precise application of activation solution, a mesh screen forsupporting the resin beads and allowing fluids to flow through, anagitator for mixing the beads as the fluids flow through, and outletsbeneath the screen for draining fluids. Components of the apparatus canbe made of stainless steel or other resilient material. In operation,resin beads in a buffer solution are poured into the vessel before beingactivated by dispersion of CNBr and acetonitrile via the dispersiontube. The beads are stirred using the agitator for a short period oftime (approximately 3 minutes or less), before the fluid is drainedthrough the outlet. The beads may be washed with water or other buffersbefore adding a coupling solution and monoclonal antibodies. The beadsand antibodies are incubated with agitation for a set period of time,while the device maintains a low temperature and monitors pH of thesolution. The antibody-coupled resin is captured on the screen andremoved from the device for use. The device can be used for a variety ofresin and antibody types. The system allows large volumes of resin to beactivated quickly and evenly, without breaking the resin beads andavoiding cross-linkage between beads. This provides more effectiveresin-antibody coupling while controlling the chemistry to lengthen theusable life of the resin beads.

A key aspect of the device is the dispersion tube, which is optimizedfor dispersion of a CNBr activation solution to the resin. This allowseven distribution of CNBr under a controlled time period and helps toachieve the addition of the activation solution quickly, preferablyunder about 3 minutes. These factors are important for the homogeneouscoupling of the antibodies and resin because they reduce variation inthe addition of solution to the resin/buffer mixture, which cantransiently exceed the buffering capacity of the buffer. Suchvariability would result in undesired iso-urea cross-linkage that wouldhydrolyze and increase antibody leaching or potentially exchange withother nucleophiles to foul the resin or cause increased leaching.Controlling the timing of activation solution helps with downstreamprocess control for antibody coupling, because it allows optimal resinactivation followed by rapid addition of antibody prior to the loss ofsignificant numbers of activated groups on the resin.

In certain aspects, the disclosure relates to a device for couplingantibodies to resin. The device includes a mixing vessel divided into anupper portion and a lower portion by a mesh screen stretched across thevessel, the upper portion having at least one inlet and the lowerportion having at least one outlet, and the mesh screen having a poresize between 5 and 80 μm. The device further includes an agitatordisposed within the upper portion and a dispersion apparatus above theagitator. The dispersion apparatus includes an elongated tubularstructure forming a lumen. The tubular structure has a proximal portionextending vertically outside of the mixing vessel with an inlet thatopens upward, a distal portion with a closed end and a plurality ofdownward-facing holes positioned horizontally within the upper portion,and an elbow connecting the distal portion and the proximal portion.

The agitator may include a rotor and a rotating impeller comprising ahub and at least two blades extending in opposite directionsperpendicularly from the hub. Each blade has an axis approximatelytransverse to the axis of the rotor upon which the hub is configured torotate. The agitator is configured to rotate to impart a force on afluid in the vessel to provide lift and to keep the beads moving acrossthe screen. The screen may also have a support beam configured tosupport the screen and prevent the screen from bowing. The screen may beconfigured to be removable from the vessel.

The agitator rotates at a speed sufficient to rapidly mix resin beadswithout breaking them. The agitator rotating speed may be between about10 and 50 RPM, and in particular embodiments the speed is 20 or 35 RPM.In embodiments, the blades have a fixed pitch and rounded edges.

In related aspects, the disclosure relates to a dispersion tubeapparatus that includes an elongated tubular structure forming a lumenwith a circular cross-section. The tubular structure includes a proximalportion with an upward-facing inlet, a distal portion with a closed endand between 4 and 100—and preferably about 8 to 30—downward-facing holesarranged in two or more rows parallel to the axis of the distal portion.The rows are positioned between about 15 and about 60 degrees apart fromeach other on the distal portion. The tubular structure also includes anelbow between the distal portion and the proximal portion, whichcomprises a bend in the tubular structure to orient the inlet such thatit opens in a direction that is substantially perpendicular to the axisof the distal portion.

In some embodiments, the distal portion of the dispersion apparatus hasthree parallel rows of downward-facing holes. In some embodiments, thereare between 12 and 25 downward-facing holes, and in a preferredembodiment there are 21 holes, with rows of 10, 8, and 3 holes. In someembodiments, the rows have different numbers of holes, and in othersthey have the same number of holes. The rows of holes may be positionedbetween about 15 and 105 degrees apart. The bend in the elbow portionorients the distal portion and the proximal portion at between 60 and120 degrees with respect to each other, and in some embodiments about80-100 degrees, and in a preferred embodiment about 90 degrees. Thetubular structure can be made of stainless steel. The dispersionapparatus may also include a valve for opening and closing the inlet anda funnel connectable to the inlet, for holding the dispersion fluidprior to use.

In a related aspect, the disclosure relates to methods for activating aresin. The method involves inserting resin beads, suspended in waterinto a mixing vessel, wherein the mixing vessel includes a dispersionapparatus, a mesh screen with holes smaller than the resin beads, and anagitator disposed above the mesh screen. The resin beads may compriseagarose, such as CL-4B or CL-2B beads or the agarose beads sold underthe trademark CAPTO by GE Healthcare Life Sciences (Marlborough, Mass.).The method further includes dispersing an activation solution comprisingCNBr and acetonitrile onto the resin beads via the dispersion apparatusand stirring the agitator for less than 5 minutes. The method theninvolves draining the activation solution through the mesh screen,thereby leaving activated resin beads supported on the screen.

In some embodiments, the method also involves washing the activatedresin beads with a fluid. Washing may include filling the mixing vesselwith the fluid, incubating while stirring the agitator, and draining thefluid from the mixing vessel. The fluid may be a buffer, water, orsolution comprising sodium bicarbonate and sodium chloride. Stirring maybe between 10 RPM and 40 RPM, and preferably about 20 RPM or 35 RPM.Incubating with stirring may last less than 4 minutes, and preferablyless than 3 minutes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C show views of the mixing vessel. FIG. 1A shows a sidecross-sectional view; FIG. 1B shows a top cross-sectional view; and FIG.1C shows a front cross-sectional view.

FIGS. 2A-G show the dispersion tube and a preferred arrangement of holesfor the dispersion tube. FIG. 2A shows a side view of the dispersiontube. FIG. 2B show a side view of the dispersion tube with a particularconfiguration of holes. FIG. 2C shows a radial cross-section of thedispersion tube, showing the angle at which the holes are drilled. FIG.2D shows a side view of the dispersion tube with the locations of a rowof 8 holes. FIG. 2E shows a radial cross-section of the dispersion tube,showing the angle at which the holes of FIG. 2D are drilled. FIG. 2Fshows a side view of the dispersion tube with the locations of row of 10holes. FIG. 2G shows a radial cross-section of the dispersion tube,showing the angle at which the holes of FIG. 2F are drilled.

FIG. 3 shows the detachable holding vessel or funnel for the activationsolution, and a valve connecting the funnel to the dispersion tube.

FIGS. 4A-C show views of the agitator. FIG. 4A is a side cross-sectionalview of the agitator. FIG. 4B is an upside-down perspective view of theagitator and blades. FIG. 4C is a close-up view of the hub and blade ofFIG. 4B.

DETAILED DESCRIPTION

Devices for isolating and purifying molecules from culture media areintegral to drug manufacturing. Many drugs involve recombinant proteinsthat are grown in culture and must then be purified and extracted fromthe media. Purification of these compounds requires precise control ofreagents and materials, and specialized equipment. Due to the complexityof preparing and using these materials, devices are necessary forgenerating purification matrices for extracting the drugs in a moreefficient, accurate, and cost-effective way. Such devices improveavailability of recombinant proteins, which are needed to treat avariety of diseases.

Many drugs involve recombinant proteins that deliver a desiredbiochemical response when introduced to a patient. For example,hemophilia drugs such as RECOMBINATE™ and ADVATE® are intravenouslyinjectable factor VIII molecules, which improve blood clotting inpatients with hemophilia to control and prevent bleeding episodes.Treatment with these drugs normalizes clotting time over the effectivedosing period by increasing plasma levels of factor VIII to temporarilycorrect the coagulation defect in those patients. Factor VIIIreplacement drugs can be used for routine prevention and reduction ofbleeding, or they can be administered before, during, and after surgeryto manage blood clotting. The goal of such treatments is to maintain aplasma factor VIII activity level at or above desired levels. Forexample, for minor bleeding episodes such as early hemarthrosis, mildmuscle bleeding, or mild oral bleeding episodes, it may be desirable toadminister a dose to achieve about 20-40% of normal factor VIII level.For moderate bleeding, such as muscle bleeding, bleeding into the oralcavity, definite hemarthrosis, and known trauma, 30-60% of normal factorVIII activity may be required. To treat major bleeding, such assignificant gastrointestinal bleeding, intracranial, intra-abdominal orintrathoracic bleeding, central nervous system bleeding, bleeding in theretropharyngeal or retroperitoneal spaces or iliopsoas sheath,fractures, or head trauma, 60-100% factor VIII level may be required.

RECOMBINATE™, ADVATE®, ADYNOVATE®, HEMOFIL®, and OBIZUR® are examples ofrecombinant factor VIII molecules that can be used to temporarilyreplace the patient's missing factor VIII to achieve hemostasis.ADVATE®, for example, is a purified glycoprotein consisting of 2,332amino acids synthesized by a genetically engineered Chinese hamsterovary cell line. ADYNOVATE® is the purified ADVATE® molecule which hasbeen covalently conjugated with one or more molecules of polyethyleneglycol, which reduces binding to the physiological factor VIII clearancereceptor (LRP1) and exhibits an extended terminal half-life. OBIZUR® isa recombinant analogue of porcine factor VIII. The B-domain present innaturally occurring porcine factor VIII is replaced with a twenty-fouramino acid linker. Once activated, the resulting drug has a comparableactivity to the endogenous human factor VIII.

To manufacture these drugs—and other drugs that involve recombinantproteins—the molecules must be purified from culture media. Generally,the recombinant cell lines express the factor VIII protein and secreteit into the cell culture medium. The molecule is then purified from theculture medium. The purification process may involve introducing themedium to one or more immunoaffinity chromatography columns in which apurification matrix, prepared by immobilization of a monoclonal antibodyto a resin, selectively isolates the factor VIII. The method may alsoinvolve one or more filtration steps. HEMOFIL®, for example, is a factorVIII protein isolated from pooled human plasma by immunoaffinitychromatography using a mouse monoclonal antibody to factor VIII,followed by an ion exchange chromatography step for furtherpurification.

Monoclonal antibodies are particularly useful for capturing factor VIIIand other target molecules. The purification matrix that selectivelypurifies the target molecule is made by coupling antibodies to a resin.Preparing the resin and coupling the antibodies is difficult becauseslight changes in the chemistry affect the bonding process and theresultant purification matrix. Devices disclosed herein provide improvedbonding of antibodies to resin beads. The chemistry of the resincoupling process is controlled to generate antibody-resin complexes withgreater efficacy and longevity. With proper manufacturing and handling,the resin can last for several years and be reused hundreds of timesbefore needing to be replaced. Because resin beads may cost tens ofthousands of dollars per liter, it is important for manufacturers tohave a reliable process for producing long-lasting antibody-resincomplexes without needlessly wasting quantities of the resin.

As stated above, small aberrations in the chemistry can reduce thefunction of the resin product. The resin must be prepared quickly andprecisely to achieve a homogenous product on a large scale. Thedisclosed devices allow a rapid homogenous reaction that is repeatableand yields a standardized product. Without such devices, the resultingresin product would have micro-heterogeneities that create leachingissues in the resin. If the resin is not homogenously activated, thebeads may become cross-linked to each other, creating clumps in theresin. Clumping and heterogeneity in the resin leads to packing problemsin the downstream purification protocols. For example, resin that clumpstogether may be less permeable to fluids passing therethrough, and causea buildup of pressure in the column. Heterogeneous resin may lead toinefficient capture of the target molecule or it may simply be unusableand need to be discarded.

The disclosed devices also prevent excess breakage of the beads duringthe processing steps. If more than about 2 percent of beads are broken,the resulting resin would be too dense and would over-compress whenloaded into the column. Broken beads therefore cause back pressure inthe column. If too much of the resin is broken during manufacture, partor all of the resin would need to be discarded, leading to excessivewaste and expense. The devices disclosed herein avoid that problem andothers. The devices optimize the resin preparation to ensure properpacking of resin in the column downstream.

In addition to improved column fluid dynamics, there are additionalbenefits to coupling resin using the disclosed apparatus. The apparatusallows greater reproducibility of resin than prior art devices. Theresulting resin is more uniform in terms of ligand density, beadcross-linking, and bead integrity. This provides greater stability,reduction of variation in the manufacturing process, and greater yield.The result is a product with a longer usable life, which can be used fora greater number of production cycles without antibody/ligand leachingand loss of binding capacity. This reduces the cost of production of theenzyme or other biologic product.

The devices can be used for many types of coupling chemistry. Forantibodies, the coupling is often amine-based. Antibodies may have 20-30amino groups and another 20-30 carboxyl groups. The coupling technologydisclosed herein can be used for any antibody or even smaller moleculecoupling. In some embodiments monoclonal antibodies are coupled, and inother embodiments polyclonal antibodies are coupled to resin. Thetechnology may be used to couple peptides as well. In any event, thegoal of the technology is to achieve homogenous coupling of a molecule(antibody, peptide, or other) to resin beads. Homogenously linking themolecules to resin gives the resultant product predictable leachingbehaviors and longer usability.

The coupling device shown in the accompanying figures quickly andeffectively activates the resin and couples antibodies, to reduce waste,improve coupling efficiency and drug recovery. As described above, thedevice generally includes a mixing vessel, a dispersion tube, and anagitator and screen.

FIGS. 1A-C show the coupling apparatus 100. FIG. 1A shows a sidecross-sectional view; FIG. 1B shows a top cross-sectional view; and FIG.1C shows a front cross-sectional view. The apparatus 100 includes amixing vessel 110, which houses the other elements described below andserves as mixing container for the various mixing steps. The vessel 110has multiple inlets 120 and outlets 130. There is a hinged hatch 150 atthe top of the vessel 110 where the resin beads may be inserted. Theresin beads may be agarose beads, such as CAPTO™, CL-4B, or CL-2B beads,available from GE Healthcare Life Sciences (Marlborough, Mass.) and aregenerally inserted into the vessel 110 in a water suspension. Up to 50liters of resin beads—and commonly about 22-39 liters—can be activatedat a time in the vessel 110. Once inside the vessel 110, the beads aresupported by a screen 310, which is made of a mesh material with holesof approximately 10-80 μm, which are small enough to prevent the resinbeads from going into the lower portion of the vessel 110 in a preferredembodiment, the holes are about 30 μm. During the activation andcoupling process, the beads are mixed by an agitator 410 (shown in FIGS.4A-C), which prevents the beads from settling on the screen and helps tomaintain homogeneity.

The vessel contains a dispersion tube 210, which is described in greaterdetail in FIGS. 2A-G, through which an activation solution of CNBr andacetonitrile is added to the beads. The dispersion tube 210 is designedto evenly distribute the activation solution over the beads while theagitator 410 keeps them moving to prevent resin clumping. As describedbelow, the agitator 410 rotates at a speed that is sufficient to providelift to the beads and to keep them moving, but gentle enough to preventbreakage of the beads. After activation, the activation solution can bedrained through the drainage spout 130, leaving the activated beads onthe screen 310.

Various fluids and buffers can be inserted into the vessel 110 via thedispersion tube 210, the inlet 120, or the hatch 150, as desired.Different solutions are required for the activation process, washingprocess, and coupling process. An exemplary resin coupling processbegins with resin being poured into the vessel 110 through the hatch150. The resin includes resin beads in a water suspension. An activationsolution of CNBr and acetonitrile is dispersed onto the resin throughthe dispersion tube 210 while the agitator 410 mixes the resin, keepingthe beads moving above the screen 310. A small amount of acetonitrilemay be used to chase the activation solution in the dispersion tube. Thebeads may be washed with the activation solution for up to 5 minutes toactivate the resin. Preferably, the activation takes place in less than3 minutes.

Various buffers and coupling solutions may be added, along withantibodies suspended in a coupling solution to couple them to the beads.With all washing and coupling steps, as during activation, the agitatorrotates to maintain constant movement of the beads, preventing them fromsticking to the screen, and to evenly distribute the various fluids andbuffers and to ensure that all surfaces of the beads are contacted.Washing steps and other mixing steps may be continuous, with fluidcontinuously flowing into the inlets and out of the outlets, or thevessel may be filled and mixing can occur for a set period of time, fromseveral seconds, to minutes, or even multiple hours. The vessel 110 alsocontains temperature controls so that the contents can be incubated at adesired temperature during mixing. The apparatus 100 may also includeone or more probes 190, such as a temperature gauge and/or a pH probe tomonitor the conditions inside the vessel during operation.

The screen 310 is designed to be removable and is held in place by aseries of clamps 330. The vessel 110 also includes trunnions 175 so thatthe apparatus can be mounted on a pivot. This allows the apparatus to betilted so that a user can fill the vessel or pour liquids out of it asneeded. The tilting function allows the processed resin to be poured outof the hatch rather than scooped manually, which prevents additionalstress on the beads and the screen. The trunnions 175 can be locked inplace during use so that it remains stationary while mixing. It isimportant for the screen 310 and agitator 410 to remain substantiallyhorizontal during mixing to achieve proper movement of the beads in thevessel and to ensure the beads are evenly exposed to the various fluidmixtures.

FIGS. 2A-G show the dispersion tube 210. As shown in FIG. 2A, thedispersion tube 210 is generally a hollow pipe with a long horizontalportion 230, a vertical portion 220, and an elbow 240. The horizontalportion 230 is generally a length of metal tubing punctuated with holesat regular or semi-regular intervals. The length of the horizontalportion 230 stretches across the vessel. In the embodiment shown, thedispersion tube 210 has a plurality of holes 231, whose positions aremarked (in FIGS. 2B, 2D, and 2F) by their distance down the length ofthe tube 210. The holes 231 allow a CNBr activation mixture to flowthrough. Together, they provide a reproducible homogeneous distributionof the activation mixture onto the resin below. The dispersion tube 210,particularly in combination with the agitator 410, preventsinconsistency in the distribution of CNBr and avoids localized pH spikesthat can lead to incorrect coupling chemistry and increased ligandleaching.

The vertical portion 220 of the dispersion tube 210 is configured toextend out of the vessel 110 while the horizontal portion 230 ispositioned within the vessel 110 above the agitator 410 and screen 310.The vertical portion 220 includes an inlet 225, through which the CNBrcan be flowed and introduced into the mixing vessel 110. The horizontalportion 230 has a closed end 235 and a plurality of holes 231. Althoughone possible arrangement of holes is shown in FIGS. 2B-G, other similararrangements of holes are contemplated as well. The holes should bedistributed along the horizontal portion of the dispersion tube toachieve sufficient coverage of the resin that is being mixed below.

FIGS. 2B-C show the locations of three holes drilled along the bottomedge of the horizontal portion 230. FIG. 2B shows a radial cross-sectionof the dispersion tube. As depicted, the holes are located at 11.75inches, 15.75 inches, and 26.50 inches measured from the axis 229 of thevertical portion 220. FIG. 2C shows a transverse cross-section of thehorizontal portion. As shown in FIG. 2C, the holes are drilled throughthe bottom point in the horizontal portion.

FIGS. 2D-E show the locations of eight holes drilled at a 45 degreeangle from the bottom edge of the horizontal portion 230. FIG. 2D showsa radial cross-section of the dispersion tube, showing the measurementsof the holes from the axis 229 of the vertical portion 220. As depicted,the holes are located at 3.00 inches, 4.00 inches, 6.00 inches, 8.00inches, 19.50 inches, 21.50 inches, 23.50 inches, and 25.00 inches. FIG.2E shows a transverse cross-section of the horizontal portion,indicating the angle at which this set of holes is drilled.

FIGS. 2F-G show the locations of another ten holes drilled at a 45degree angle, on the other side of the dispersion tube 210 from the setof eight holes (shown in FIG. 2D). FIG. 2F shows a radial cross-sectionof the dispersion tube, showing the measurements of the holes from theaxis 229 of the vertical portion 220. As depicted, the holes are locatedat 2.50 inches, 3.50 inches, 4.50 inches, 6.50 inches, 9.50 inches,11.50 inches, 21.00 inches, 23.00 inches, 24.00 inches, and 26.00inches. FIG. 2G shows a transverse cross-section of the horizontalportion, indicating the angle at which this set of holes is drilled.

As depicted in FIGS. 2B-G, the sets of holes are arranged in threeparallel rows drilled into the bottom half of the horizontal portion230. Each of the three rows has a different number of holes 231, andeach hole is drilled at a distance along the horizontal portion 230 thatis different from the distance of all other holes. The location of theholes is an important consideration for ensuring even distribution ofthe activation solution onto the resin. This arrangement of holes wasfound to be optimal based on salt modeling experiments. However, otherarrangements of holes may also be used as well. The precise number ofholes, their relation to each other, the number of rows, the angle atwhich the holes are drilled, the size of the holes, and other factorscan be adjusted as needed.

Adding the activation mixture using the dispersion tube is superior tomanual addition because it achieves greater uniformity in the resultantresin product. As explained above, resin manufactured by the disclosedcoupling apparatus functions differently in the column as compared withresin generated using other methods. Using the dispersion tube can add ahundred or more cycles to the usable life of the resin beads.

Another important benefit of the dispersion tube is that it allowshomogenous distribution of the activation mixture in a very short periodof time. Activation of the resin with CNBr needs to occur quickly inorder to reduce the occurrence of cross-links. In most cases it shouldbe completed in less than 5 minutes, and ideally it should be completedin less than about 3 minutes. If the activation process takessignificantly longer than 3 minutes, coupling efficiency is reducedresulting in excessive cross-linkage. The dispersion tube, particularlyin conjunction with the agitator system described below, allows theactivation process to occur quickly in order to produce a high qualityresin. The CNBr can be added to up to about 40 liters of resin, mixed,and removed in under 3 minutes, and in some cases under 2 minutes.

FIG. 3 shows a detachable holding vessel or funnel 290 for theCNBr-acetonitrile solution, which can be included in the apparatus. Theoutlet 291 of the funnel 290 is in fluid communication with the inlet225 on the vertical portion 220 of the dispersion tube. A valve 280controls flow of the fluid from the funnel 290 into the dispersion tube210. In use, the detachable holding vessel 290 is filled with the CNBrsolution and attached to the dispersion tube. The removability of thefunnel 290 allows the CNBr to be more safely handled and avoid spillage.CNBr-acetonitrile mixture is corrosive and dangerous, and so theremovable funnel allows the mixture to be mixed under a hood beforebeing connected to the coupling apparatus.

Once the resin is prepared for activation, the valve 280 is opened toallow the solution to travel through the dispersion tube 210 and ontothe resin. The dispersion typically takes 2-4 minutes. The solution canbe chased with a small volume of acetonitrile solution to rinseremaining CNBr out of the funnel 290 and tube 210.

Inside the vessel 110, the resin beads are stirred by the agitator 410,which keeps them moving across the screen 310. A side cross-sectionalview of the agitator is shown in FIG. 4A. The agitator includes a rotor420, which is a vertical rod attached to a stirring motor (not shown).The agitator also includes blades 440 attached to the rod at a hub 445.In FIG. 4A, the blades 440 are shown in cross-section. An upside-downperspective view of the agitator is shown in FIG. 4B to provide aclearer view of the blade 440 configuration. The blades 440 rotate tolift the resin beads off of the screen 310. The agitator may alsoinclude a circular frame 450 that circumscribes the outermost points ofthe blades 440, and it may further include spokes 460 for additionalsupport. Referring again to FIG. 4A, the bottom edge of the frame 450 isconfigured to be parallel with the screen (not shown), which is disposedbeneath the agitator 410.

The blades 440 shown in FIG. 4A have a width of 3.0 inches, a thicknessof 0.25 inches, and a length of 28.0 inches. However, the dimensions ofthe blades 440 may be different for different uses. In some embodimentsthe agitator 410 may include more than two blades. As shown, each blade440 has a pitch of 40 degrees with respect to the horizontal. Indifferent embodiments, the blades may have different pitch angles, suchas 10 degrees, 20 degrees, 30 degrees, 50 degrees, 60 degrees, 70degrees, 80 degrees, and the like. The blades 440 can have rounded edges449 as shown in FIG. 4C, which helps to prevent bead breakage. Theagitator may be made of stainless steel or another resilient material.In the embodiment shown in FIGS. 4A-C, the agitator is configured torotate clockwise so that the blades create an upward lift in the fluidin the vessel (rather than pushing the fluid downward into the screen).

The rotational speed of the agitator can be adjusted based on the typeof resin beads that are being used and the amount of mixing needed. Theagitator must provide enough lift to keep the beads suspended and movingacross the screen, and to rapidly mix them while avoiding breaking orcrushing the beads. Since different beads settle at different rates, theagitator speed should be adjustable to account for different types ofbeads. For example, CAPTO™ beads settle faster than CL-4B or CL-2Bbeads. In various embodiments the agitator may rotate at up to 60 RPM. Apreferred agitation speed is 35 RPM. Another preferred agitation speedis 20 RPM. The speed may be determined based on the type of bead, thetype of liquid, the temperature of liquid, and the length of incubationtime. Based on these factors, the agitator is designed to mix the resinthoroughly during the resin activation process and during the antibodycoupling process. This allows correct mixing for chemical dispersion andavoids resin bead crushing/breaking during the coupling process. Theagitator also increases the resin recovery rate because it prevents theresin from sticking to the screen.

In some embodiments, the two blades 440 of the agitator may be angled inopposite directions to provide for a dynamic resin agitation andsuspension within the solution inside the vessel. In other words, oneblade is angled downward causing the resin to be patted down onto thescreen, while the other is angled upward to create lift. In otherembodiments, the agitator blades are both angled the same direction. Theagitator is configured to create a flow pattern that causes adequatemixing and suspension of the resin slurry when the agitator is rotated.

In the resin activation process, while the beads are moving, thedispersion tube 210 described above can be used to distribute activationmixture onto the beads. According to various methods, buffers and otherfluids can be pumped through the vessel during the activation, washing,and coupling process. The fluids pass through the screen and drain outthrough an outlet at the bottom of the vessel, while the agitator 410keeps the beads moving above the screen.

The screen shown in FIGS. 1A-C has a 30 inch diameter with a mesh poresize of 30 μm to retain the resin and allow the different solutions andbuffers to be filtered through the mesh during the coupling process. Themesh may have a pore size as small as about 5 μm and as up to a maximumof about 80 μm. In any case the pore size should be small enough thatbeads cannot get through the screen. The screen may be a single meshscreen, or it may be two, three, or more sintered layers. Topmesh TM3-BM30 is a mesh that is compatible with the present invention, supplied byG. BOPP USA, INC. (Wappingers Falls, N.Y.).

In an embodiment, the top layer is a 30 μm filtration mesh, sinteredwith a 0.850 mm square mesh and a 2.0 mm square mesh for support. Thescreen is welded to a stainless steel 30 inch frame. The screen may beheld in place within the vessel by a gasket, which may be removable fromthe screen.

As discussed above, the agitator is designed to sufficiently mix theresin while preventing the resin from breaking down, which would clogthe screen and prevent efficient resin recovery. The screen is removablehowever, so that it can be cleaned or replaced after normal wear andtear. The coupling activation process requires rapid buffer exchanges,on the order of minutes, where clogging of the screen will lead toslower buffer exchange rates, thus causing process non conformances andpotential rejection of the lot.

The screen is resilient and resistant to tearing or breaking, to preventresin from escaping through the screen. In addition, the screenstructure is strong enough to not bow or bend under the weight of theresin. Bowing would lead to non-homogenous mixing or poor buffer flowthrough the apparatus and may cause clogging of the mesh. Bowing couldalso block the release valve 130 for the vessel 110 which is located ashort distance beneath the level of the screen. In some embodiments aperforated support plate may be positioned between the layers for addedrigidity and durability. Additionally or alternatively the screen mayinclude a stainless steel support beam positioned underneath the screento support the center. Inclusion of support structures helps to preventheterogeneity in the distribution of the beads across the screen. Unevendistribution would lead to resin clumping and changes in the chemistryand the leaching properties of the resin.

INCORPORATION BY REFERENCE

Any and all references and citations to other documents, such aspatents, patent applications, patent publications, journals, books,papers, and web contents, which have been made throughout thisdisclosure, are hereby incorporated herein by reference in theirentirety for all purposes.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein.

What is claimed is:
 1. A dispersion apparatus comprising: an elongatedtubular structure forming a lumen having a circular cross-section, thetubular structure comprising: (a) a proximal portion with anupward-facing inlet; (b) a distal portion with a closed end, the distalportion comprising a plurality of downward-facing holes arranged inthree rows parallel to the axis of the distal portion, wherein the threerows are positioned between about 15 and about 60 degrees apart fromeach other on the distal portion, and wherein all holes of the distalportion are positioned facing a mesh screen placed below the elongatedtubular structure; and (c) an elbow between the distal portion and theproximal portion, the elbow comprising a bend in the tubular structurewhich orients the inlet such that it opens in a direction that issubstantially perpendicular from the axis of the distal portion.
 2. Thedispersion apparatus of claim 1, wherein the distal portion comprisesbetween 8 and 30 downward-facing holes.
 3. The dispersion apparatus ofclaim 2, wherein the distal portion comprises exactly 21 downward-facingholes.
 4. The dispersion apparatus of claim 3, wherein a first of thethree rows has 10 holes, a second of the three rows has 8 holes, and athird of the three rows has 3 holes.
 5. The dispersion apparatus ofclaim 1, wherein the three rows have different numbers of holes.
 6. Thedispersion apparatus of claim 1, wherein the three rows have the samenumber of holes.
 7. The dispersion apparatus of claim 1, wherein thethree rows are each positioned between 30 and 60 degrees apart.
 8. Thedispersion apparatus of claim 7, wherein the three rows are eachpositioned 45 degrees apart.
 9. The dispersion apparatus of claim 1,wherein the bend in the elbow orients the distal portion and theproximal portion at between 60 and 120 degrees with respect to eachother.
 10. The dispersion apparatus of claim 9, wherein the bend in theelbow orients the distal portion and the proximal portion at between 80and 100 degrees with respect to each other.
 11. The dispersion apparatusof claim 10, wherein the bend in the elbow orients the distal portionand the proximal portion exactly 90 degrees with respect to each other.12. The dispersion apparatus of claim 1, wherein the tubular structurecomprises stainless steel.
 13. The dispersion apparatus of claim 1,further comprising a valve for opening and closing the inlet.
 14. Thedispersion apparatus of claim 1, further comprising a funnel connectableto the inlet.